The long term goal of this investigator is a molecular dissection of the assembly and trafficking of human and murine MHC Class II molecules in vitro and in living cells. By preparing semi-intact cell preparations and cell-free systems, the aim is to duplicate assembly and sorting events in vitro. Translation in vitro of HLA Class II alpha and beta mRNAs, in the presence and absence of invariant chain (Ii) mRNA will be performed in membrane-supplemented translation systems; the formation of proper Class II oligomers will be assessed immunochemically and biochemically. The identity of the proteins involved in sorting will be investigated in semi-intact cells and in cell-free systems, using pharmacological agents and affinity purification techniques. The Ii chain is removed by the combined effects of acidic pH and proteolytic activity in the endocytic pathway to render the resulting Class II heterodimers capable of peptide binding and to release them for transport to the cell surface. The proteases involved in Ii breakdown are also likely candidates for the enzymes that generate the peptides that bind to Class II molecules. The lysosomal proteases cathepsin B, D and E, have been implicated in this process. The role of these proteases in Class II biosynthesis and antigen processing will be assessed by constructing strains of mice deficient in them. The resultant mice will be used as a source of antigen-presenting cells to examine the precise role of cathespins B, D and E in antigen processing. It is anticipated that cathepsin-deficient mice may show aberrations both in the MHC Class II restricted presentation of certain sets of peptides and in the levels of Class II expression. Because the peptides bound by Class II molecules are involved in positive and negative selection, the diversity and functionality of the T cell repertoire as it emerges in cathepsin deficient mice will be examined in detail.
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